Our goal is to understand the mechanism by which neurotransmitter transport proteins move small molecules and ions across biological membranes. In recent years it has become apparent that many families of transport proteins are related at the structural level. The proteins are responsible for transporting a wide variety of molecules. Within the family containing neurotransmitter transporters that are targets of cocaine and amphetamines, a bacterial protein (LeuT) has been crystallized in complex with several substrates and drugs. These crystal structures have provided dramatic new insight into these proteins and have led to several proposals concerning transport mechanism. These transporter families are all based on a protein fold containing two structurally similar repeats in topologicaly opposite orientations relative to the membrane. Analysis of this structure suggests that it is composed of a scaffold and a 4-helix bundle that, by tilting at different angles relative to the scaffold, can expose the central binding site either to one side of the membrane or the other. Both the bundle and the scaffold are composed of elements from each of the two repeats. We have proposed a mechanism to explain the function of all these structurally related transport proteins. In this mechanism, the bundle rocks back and forth between positions that alternately create pathways from each side of the membrane through which substrate and ions access the binding site. The specific aims of this project are to use biochemical approaches to test the validity of the rocking bundle and other proposed mechanisms for transport. We will focus on the pathways through which serotonin and dopamine travel through their respective transporters as they cross the membrane. PUBLIC HEALTH RELEVANCE: The movement of molecules across cell membranes is critical for many important physiological processes including the uptake of neurotransmitters in the brain, a process that is affected by psychostimulant drugs like cocaine and amphetamines. The molecular structures of transporter proteins responsible for these and many other transport reactions have recently been found to be closely related. Our goal, to reveal how these related proteins work, will allow a better understanding of the physiological processes that depend on them and provide a basis for therapy to treat related disorders.